This study,
performed by Dr. A. CuneytTas, reports the rapid formation of apatitic calcium phosphate coatings on Ti6Al4V using high
ionic strength solutions compared to synthetic/simulated body fluids (SBFs).

The
super-strength solution developed in this study has ten times the
concentration of calcium and phosphate ions as compared to conventional SBF,
and is referred to as 10 x SBF.

The idea is to
significantly enhance the rate of coating formation, which requires 1 to 3
weeks of immersion time using conventional 1.5 X SBF.

The interesting
features of the present technique are given in the following. First, the
solutions do not need any buffering agents. Given the short duration of coating
period, these are not really needed.

Second,
during the process homogeneous precipitation of nano-clusters
took place. However, their presence did not adversely affect the coating
process.

Third,
other than simple surface treatments to begin with, no other additional
intermediate steps were necessary. The only step needed after the preparation
of the solution from reagents is the addition of proper amounts of NaHCO3
just prior to the coating procedure.

Fourth,
such a procedure led to a significant enhancement of coating rate enabling the
formation in as little as 2 hours.

Finally, the
adhesion strength of the coatings was comparable to coatings produced from 1.5
X SBF over a prolonged period of time.

SBF
solutions, in close resemblance to the Hanks Balanced Salt Solution (HBSS) [4],
are prepared with the aim of mimicking the ion concentrations present in the
human plasma. It is noted that physiological HBSS solutions are also able to
induce apatite formation on titanium [5].

To mimic
physiological solutions, conventional SBFs (i.e., 1,
1.5, 2, or 5 X SBF) have relatively low calcium and phosphate ion
concentrations, namely, 2.5 mM and 1.0 mM, respectively, for 1 X SBF [6]. pH
of SBF solutions was typically brought to the physiologic value of 7.4 by using
buffers, such as TRIS [3] or HEPES [7]. The buffering agent TRIS present in
conventional SBF formulations, for instance, is reported [8] to form soluble
complexes with several cations, including Ca2+,
which further reduces the concentration of free Ca2+ ions available
for coating.

The hydrogencarbonate ion (HCO3-)
concentration in conventional SBF solutions was between 4.2 mM
(equal to that of HBSS) and 27 mM [7, 9, 10].

However,
having their ionic compositions similar to that of human blood plasma, SBF
formulations have only been slightly supersaturated with respect to
precipitation of the apatitic calcium phosphates. As
a direct consequence, nucleation and precipitation of calcium phosphates from SBFs are rather slow [11]. To get total surface coverage of
a 10 x 10 x 1 mm titanium or titanium alloy substrate immersed into a 1.5x SBF
solution, one typically needs to wait for 2 to 3 weeks, with frequent
replenishment of the solution [12].

The motivation
in this work is to enhance the kinetics of coating deposition. This enhanced
kinetic should do away with the necessity of using buffers.

In order
to achieve the above objective, Barrereet al.
[13-17] have recently developed unique 5 X SBF-like solution recipes (with pH
values close to 5.8), which did not employ any buffering agent, such as TRIS or
HEPES. In these studies [13-17], coating was achieved by employing two
different solutions (solutions A and B as they referred), and pH was adjusted by
bubbling CO2 gas into the reaction chamber. A coating thickness of
about 30 mm was achieved
only after 6 h of immersion. However, they also introduced additional
intermediate steps. These included [13] immersing the metal strips in the first
solution (to seed the surface with calcium phosphate nuclei) for 24 h at 37°C, followed by another soaking in
their second solution (to form the actual coat layers) for 6 to 48 h at 50°C [13]. These additional
intermediate steps add extra time and opposes the advantage gained by the
enhanced kinetics.

The aim
of this study was to present the preparation of a new acidic solution, which
contains 10 times the calcium and phosphate ion concentrations of human blood
plasma. Such a solution should enhance the kinetics of coating formation even
more. Further it is preferred that other than the surface treatment step, not
too many intermediate steps are involved. The only step that is needed is to
add NaHCO3 into the solution to raise its pH to around 6.5.

The
resultant solution is able to coat Ti6Al4V strips for the first time (RT: 22±1°C)
rapidly, in as little as 2
hours. It is shown that it is not necessary to use biomimetic
conditions for coating purposes.

Preparation of Ti6Al4V strips

Sheets of Ti6Al4V (Goodfellow) were cut into
rectangular strips with typical dimensions of 10 x 10 x 0.20 mm and first
abraded manually with a 1200-grit SiC paper. Strips
were then cleaned with acetone (15 min), ethanol (15 min) and deionized water (rinsing), followed by etching each strip
in 150 mL of a 5 M KOH solution at 60°C for 24 h, in a sealed
glass bottle. Thoroughly rinsed (w/water) strips were finally heat-treated at
600°C
for 1 h in Al2O3 boats, with heating and cooling rates of
3°C/min.

Coating solutions

Solution preparation recipe (for a total aqueous volume
of 2 L) is given below in Table 1. The chemicals given in Table 1 are added, in
the order written, to 1900 mL of deionized
water in a glass beaker of 3.5 L-capacity. Before the addition of the next
chemical, the previous one was completely dissolved in water. After all the
reagents were dissolved at RT, the solution was made up to 2 L by adding proper
amount of water. This stable stock solution of pH value of 4.35-4.40 can be
stored at RT, in a capped glass bottle, for several months without
precipitation.

Table 1 Solution
preparation recipe, for a total volume of 2 L

________________________________________________________________

Reagent
Order Amount
(g) Concentration (mM)

________________________________________________________________

NaCl
1
116.8860
1000

KCl
2
0.7456 5

CaCl2.2H2O
3
7.3508
25

MgCl2.6H2O
4
2.0330
5

NaH2PO4
5
2.3996
10

________________________________________________________________

Just prior to coating
a Ti6Al4V strip, a 200 mL portion of this stock
solution was placed into a 250 mL-capacity glass
beaker, and a proper amount of NaHCO3 powder was added to raise the hydrogencarbonate ion (HCO3-)
concentration to 10 mM, under vigorous stirring.
Following the rapid dissolution of the NaHCO3, the pH of the clear
solution rose to 6.50 at RT. This solution (with an ionic strength of 1137.5 mM) was then transferred to a 250 mL-capacity
glass bottle, which contained the Ti6Al4V strip inside, tightly capped and kept
at RT for 2 to 6 hours during coating.

Characterization

After the
experiments were over, the strips were taken out of the solutions and rinsed
with an ample supply of deionized water and ethanol,
followed by drying in air. Samples were characterized by XRD (Model XDS 2000, Scintag Corp., Cu K(), FTIR (Bruker, ATR-FTIR), and SEM (Hitachi S-4700 in the secondary
electron mode, acceleration voltage 5-15 kV). Platinum sputtering was used to
render conductive surfaces that were necessary for the SEM investigations. In
order to measure the thickness of the coat layers, the strips were tilted by 45
degrees and studied by
SEM.

Results

The chemical and thermal treatment of Ti6Al4V strips prior to the
coating runs were mainly performed according to the previously published
methods [6, 18, 19]. However, in our modification to
the alkali treatment procedure, we have used 5 M KOH solution in lieu of 5 M NaOH. Figures 1a and 1b show the surface of 5 M KOH + 600°C-treated metal surface,
and the aggregated rosettes seen on the surface belong to a potassium titanate phase of an approximate composition of K2Ti5O11.

A phase of similar stoichiometry (i.e., Na2Ti5O11)
was also observed in case of using 5 M NaOH+600°C-treatment [19]. The
surface of the alkali- and heat-treated strips also contained rutile (TiO2) as a minor phase. K ions on the
surfaces of such strips, when exposed to the coating solution, are released
into the solution in exchange of H3O+ ions, and
eventually resulting in the formation of a Ti-OH layer. Ca2+ ions
from the coating solution are then incorporated in this basic layer and act as
embryonic sites for the nucleation of carbonated apatitic
calcium phosphates [18]. The dimensions of crevices or pits created on Ti6Al4V
surface in the etching step of 5 M KOH-soaking was found (Fig. 1) to be much
larger than those created in using 5 M NaOH [19].
Bigger crevices (as compared to sub-micron pits obtained with NaOH) are more suitable for the attachment of few
microns-large calcium phosphate globules.

The coating solution described above was not stable against
precipitation (at RT) after the addition of NaHCO3 to raise its pH
to the vicinity of 6.5. The stability against homogeneous precipitation only
lasted from 5 to 10 minutes at RT, following the addition of NaHCO3.

After that period, solutions containing the metal strips slowly started
to display turbidity (from 10 minutes to the end of the first hour), and by the
end of 2 hours the solution turned opaque. The colloidal precipitates formed in
the solution stay suspended, and could only be separated from the mother liquor
by centrifugal filtration (>3000 rpm).

However, it is interesting to note that the solution pH at the end of 2
hours of soaking period stayed the same or slightly increased to around 6.57 or
6.58 (®download the pH chart).That
slight increase in pH was ascribed to the release of CO2 [14]. A pH
decrease would have been encountered during the formation of colloidal
precipitates due to H+ release, but as it was reported previously
such a pH drop was not always observed [14, 20].

In order to perform a run with a 6 hours-of-total-soaking time, the
coating solution for the same strip was replenished twice with a new
transparent solution (of pH = 6.5) at the end of each 2-hours segment. The
start of precipitation indicated the stage where the solution reached supersaturation.

Figure 2 depicts the SEM photomicrographs of coated surfaces of Ti6Al4V
strips as a function of coating time (1 to 6 h; Figs. 2a through 2d) at RT.

The observed globules of apatitic calcium
phosphate were quite similar to the previously reported results relevant to biomimetic SBF coating, excepting that biomimetic
conditions were not met here. High-magnification photomicrograph of Fig. 2d
showed that the globules actually consisted of petal-like nanoclusters.
The significant extent of surface coverage of the tivanium
strip, in only 6 hours of coating, was exemplified in the macro-scale SEM
picture depicted in Fig. 2c. Cracks seen in the micrographs of the coat layers
were probably formed during the drying process of the coated samples [13]. The
adhesion of the coat layers was resistant to finger-nail scratch tests, and
there was no difference in adhesion strength as compared to 1.5 X SBF-coated
Ti6Al4V strips.

XRD data of the coated strips also confirmed the nature of these
globules, as shown in Figure 3.

These precipitates were filtered from their mother liquor by centrifugation,
washed three times with water, and once with ethanol, followed by drying at RT
overnight. The SEM micrograph of Figures 4a and 4b, and the XRD data of these
precipitates given in Fig. 5, as well as the FTIR data supplied in Fig. 6, also
indicated that the nano-crystalline, bone-like apatitic calcium phosphate formed in the solution
aggregated during filtration and drying.

Fig. 5
XRD data for the colloidal precipitates formed in coating solutions

Bands of
the O–H stretching and bending of H2O were seen at, respectively,
3440 and 1649 cm-1. Presence of carbonate groups was confirmed by
the bands at 1490-1420 and 875 cm-1. PO4 bands were
recorded at 570 and 603 (n4), 962 (n1), 1045 and 1096 (n3) cm-1. It is important to note that
neither the precipitates themselves nor the coating layer (on Ti6Al4V strips)
contained CaCO3 (calcite) [21].

Titanium or titanium alloy surfaces are coated with a carbonated and
poorly-crystalline apatitic calcium phosphate layer to
impart the metal surface a certain degree of in vivo bioactivity. If
this is the sole aim, then there is no need to maintain the pH value of a
coating solution exactly at the physiologic value of 7.4. This point has been
successfully confirmed in the work of Barrere, et
al. [13-17, 22]. One only needs to be aware of the delicate balance between
the solution pH, HCO3- ion concentration and temperature
in determining which phases will be soluble or not under a specific set of
those conditions [23]. On the other hand, the presence of TRIS or HEPES (added
for the sole purpose of fixing the solution pH at around 7.4) in a SBF
formulation simply retards the coating process to the level that in order to
obtain a decent surface coverage one needs to wait for 1 or 3 weeks [6-8, 18].

Fast coating solutions, sometimes named as supersaturated calcification
solutions (SCS) are not new either; for instance, the pioneering work of Wen, et al. [24] showed that even in a TRIS-buffered
SCS solution it would be possible to form 16 mm-thick calcium
phosphate coat layers in after 16 hours of immersion. More recently, Choi, et al., [25] reported the RT coating (about 10
mm-thick in 24 hours) of nickel-titanium
alloy surfaces by a simple SCS solution, which was not even buffered at the
physiologic pH. The present paper corroborates these previous findings and
reports further improvements.

It is known that an amorphous calcium phosphate
(ACP) precursor is always present during the precipitation of apatitic calcium phosphates from the highly supersaturated
solutions, such as the one used here [26]. Posner, et al. [27] proposed
that the process of ACP formation in solution involved the formation first of Ca9(PO4)6 clusters which then
aggregated randomly to produce the larger spherical particles or globules (as
seen in Figs. 2d and 4), with the intercluster space
filled with water. Such clusters (with a diameter of about 9.5 Angstrom [26]),
we believe, are the transient solution precursors to the formation of
carbonated globules with the stoichiometry of Ca10-x(HPO4)x(PO4)6-x(OH)2-x,
where x might be converging to 1 [14].

Onuma, et al. [28] have demonstrated, by
using dynamic light scattering, the presence of calcium phosphate clusters from
0.7 to 1.0 nm in size in clear simulated body fluids. They reported that
calcium phosphate clusters were present in SBF even when there was no
precipitation. This was true after 5 months of storage at RT. The solution
coating procedure described here probably triggered the hexagonal packing [28]
of those nanoclusters to form apatitic
calcium phosphates, just within the first 5 to 10 minutes, following the
introduction of NaHCO3 to an otherwise acidic calcium phosphate
solution.

One can also speculate besides the substrate
coating procedure outlined here that these 10 X SBF solutions may also be used
to produce carbonated, bone-like apatitic calcium
phosphate powders [9]. Following proper physiologic-temperature and pH
granulation processing, such biomimetic powders may,
for instance, lead to the manufacure of
collagen-apatite composites or self-setting orthopedic cements of high resorbability.

The use of NaHCO3 with a concentrated (10 times of Ca2+
and HPO42- ion concentrations) synthetic body fluid-like
solution of ionic strength of 1137.5 mM
allowed the formation of an apatitic calcium
phosphate layer on Ti6Al4V at room temperature within 2 to 4 hours.

The coating solutions of pH 6.5 did not necessitate the use of buffering
agents.

The surfaces of the Ti6Al4V strips were chemically etched in 5 M KOH
solution and thermally treated afterwards at 600°C, prior to soaking in
10 x SBF.

Formation of colloidal precipitates, within the solution, was observed
during the first hour of soaking at RT, but apparently the presence of those
fine precipitates did not hinder the coating process.